Functional Metagenomics Approach for Discovery of Novel Cold-Active Protease from the Antarctic Region

Abstract

The structural complexity of bacterial life makes most of it impossible to culture. Functional metagenomics approaches overcome the limitations of a culture-based approach in exploring and assessing the genetic materials of uncultured microbes. The objective of this study was to identify clones with cold-active proteases through functional metagenomics. In this work, the environmental DNA (eDNA) isolated directly from Antarctic soils was ligated into the pCC1FOS fosmid vector, transformed into EPI300-T1R E. coli host cells, and screened for proteolytic enzymes. Positive protease-producing clones were identified and isolated on skim milk agar supplemented with chloramphenicol and arabinose. This clone harbored a fosmid, pCC1FOS, which has a 48.5 kb insert that has been completely sequenced in both directions. Further analysis of the insert showed 70 NODEs. The NODE_42 encoded hypothetical protein of 297 amino acids showed a significant match to Peptidase M23 and PG-binding 1 proteins families. A three-dimensional model of the predicted protease was generated based on the known mesophilic protease of Neisseria meningitides (PDB: 3SLU). The structural alignment showed 27.07 % similarity with RMSD value of 0.402 Å based on 58 aligned residues. The active site residues were identical, but major deletions were observed in the predicted proteases. This predicted protease showed higher activity at -20 °C and 20 °C than the positive control (protease from bovine pancreas). Functional metagenomics is a promising approach in the discovery of cold-active protease with low homology to the known sequences and expressed in the host cell that has the potential for bioprospecting in low-temperature applications. ABSTRAK: Kesukaran struktur kehidupan bakteria menyebabkan kebanyakan bakteria sukar dikultur. Saringan fungsi metagenomik dapat mengatasi kekangan saringan berasaskan kultur dalam meneroka dan menilai bahan genetik mikrob tidak kultur. Objektif kajian ini adalah bagi mengenal pasti klon protease aktif sejuk melalui saringan fungsi metagenomik. Kajian ini mencadangkan DNA alam sekitar (eDNA) yang diasingkan secara langsung dari tanah Antartika, dan dimasukkan ke dalam vektor fosmid pCC1FOS, ditransformasikan ke dalam sel hos EPI300-T1R E. coli, dan disaring untuk enzim proteolitik. Klon yang menghasilkan protease ini dikenal pasti dan diasingkan daripada agar susu skim. Agar telah ditambah dengan kloramfenikol dan arabinosa. Klon ini mengandungi fosmid pCC1FOS, di mana sisipan sebesar 48.5 kb telah disusun berturut-turut sepenuhnya dari kedua-dua arah. Analisis lanjut pada sisipan ini menunjukkan terdapat 70 NODE. NODE_42 mengandungi 297 asid amino protein hipotetis, iaitu sangat serasi dengan protein Peptidase M23 dan PG-binding 1. Model tiga dimensi protease yang diramalkan ini dihasilkan berdasarkan protease mesofilik Neisseria meningitides (PDB: 3SLU). Susunan struktur menunjukkan 27.07% persamaan nilai RMSD sebanyak 0.402 Å berdasarkan 58 sisa sejajar. Sisa tapak aktif kedua-duanya adalah sama, tetapi terdapat pemotongan besar telah berlaku dalam protease ramalan. Protease ini menunjukkan aktiviti enzim lebih tinggi pada suhu -20 °C dan 20 °C berbanding kawalan positif (protease dari pankreas lembu). Saringan fungsi metagenomik berhasil menemukan protease aktif sejuk berhomologi rendah terhadap susunan yang diketahui dan diekspresi dalam sel hos. Ia berpotensi bagi bioprospeksi dalam aplikasi bersuhu rendah.

Keywords

• metagenomics
• cold-active
• enzyme
• protease
• Antarctica